The nature of the signals that are perceived as T cell activating versus tolerogenic is as yet unclear. A recently revisited readout for T cell activation is receptor clustering at the T celI-APC interface followed by coalescence of these clusters into the central "synapse". We and others have demonstrated that while initial micro-clusters are associated with the onset of signaling, a program of cellular re-polarization is necessary for the formation of the central synapse structure and for sustained signaling. All of the known signaling players in T cell activation that have been examined have been shown to coalesce at the synapse making this site a critical hub for signaling and making participation in this structure a 'biosensor' at some level for participation in signaling. In this proposal, we screen a library of T cell expressed gene-products for their participation in the immunological synapse under varying activation conditions. To do this, we will i.) Construct fluorescent-protein fusion libraries in T cells using gene-trapping and/or cDNA fusions. This technology is already partially developed in our laboratory, ii.) The library will be phenotypically screened for localization and differential localization of fusions to the synapse under varying activation conditions. For this, we will develop and utilize novel medium-high throughput imaging-based single-cell assays including instrumentation and analysis algorithms. In addition to identifying these gene products, our study will open up a technology for larger scale analyses of activating and tolerant signaling. Thus, consistent with this R21 PA, our studies will investigate the unique and innovative use of existing methodology to explore a new area and will develop novel techniques that could have a major impact on the field of biomedical research.